Tipping the balance toward transplantation tolerance: in vivo therapy using a mutated IL-2

Immune tolerance to allogenic transplanted tissues remains elusive, and therapeutics promoting CD4+FOXP3+ Tregs are required to achieve this ultimate goal. In this issue of the JCI, Efe and colleagues engineered an Fc domain fused to a human mutein IL-2 (mIL-2–Fc) bearing mutations that confer preferential binding to the high-affinity IL-2 receptor expressed on Tregs. In vivo mIL-2–Fc therapy effectively heightened mouse, monkey, and human Treg numbers, promoted tolerance to minor antigen mismatched skin grafts in mice, and synergized with immunosuppressive drugs used in the clinic. These findings warrant clinical trials that assess the efficacy of mIL-2–Fc in transplantation.

suppression (by triggering the high-affinity trimeric IL-2R on Tregs in the context of autoimmunity and transplantation).
However, given the double-edged sword aspect of IL-2 therapy, its use in the clinic can lead to unexpected outcomes.One example includes a clinical trial in which stable liver transplant recipients received low-dose IL-2 as a means to promote Tregs and reduce immunosuppression (2).In preclinical studies, low-dose IL-2 was shown to preferentially expand Tregs over effector T cells and NK cells (3).All of the patients with liver transplants who received low-dose IL-2 had elevated circulating Tregs.However, low-dose IL-2 in this clinical trial was detrimental, as the patients developed signs of rejection, leading to an early termination of the trial (2).Similar observations were reported in a clinical trial involving patients with type 1 diabetes who concomitantly received low-dose IL-2 and Treg therapy (4).Low-dose IL-2 treatment not only effectively promoted both adoptively transferred and endogenous Tregs, but also expanded cytotoxic immune cells.
On the other hand, low-dose IL2 therapy has demonstrated beneficial effects in some clinical trials for the treatment of various autoimmune disorders, more specifically in patients with systemic lupus erythematosus (5).Thus, it appears that the outcomes under low-dose IL-2 therapy may vary depending on the underlying cytotoxic immune response at the time of IL-2 treatment, with the parallel Treg expansion having an insufficient inhibitory effect on these cytotoxic immune cells.Overall, these trials demonstrate that improved therapeutics targeting specific IL-2Rs are necessary.
IL-2 muteins were developed to skew the specificity of IL-2 toward either the high-affinity trimeric or the intermediate-affinity dimeric IL-2R.To achieve such a goal, these mutated IL-2 proteins displayed a reduced affinity either for CD122 or CD25.The crystal structure of IL-2 bound to the IL-2R was helpful in identifying the amino acid residues implicated in IL-2 interactions with either CD122 or CD25.Consequently, targeted amino acid substitutions led to two versions of IL-2 mutein (no-α IL-2 and IL-2 superkine) displaying lower affinity for CD25 and thus favored dimeric IL-2R signaling on memory T cells and NK cells.In vivo therapy using either no-α IL-2 or IL-2 superkine enhanced immune responses to tumors, owing to increased CD8 + T cell responses and lower Treg expansion compared with native IL-2 therapy.IL-2 muteins with lower affinity for CD122, and thus preferential specificity for the trimeric IL-2R, were also generated Immune tolerance to allogenic transplanted tissues remains elusive, and therapeutics promoting CD4 + FOXP3 + Tregs are required to achieve this ultimate goal.In this issue of the JCI, Efe and colleagues engineered an Fc domain fused to a human mutein IL-2 (mIL-2-Fc) bearing mutations that confer preferential binding to the high-affinity IL-2 receptor expressed on Tregs.In vivo mIL-2-Fc therapy effectively heightened mouse, monkey, and human Treg numbers, promoted tolerance to minor antigen mismatched skin grafts in mice, and synergized with immunosuppressive drugs used in the clinic.These findings warrant clinical trials that assess the efficacy of mIL-2-Fc in transplantation.
mouse and human Tregs in vitro.In addition, mIL-2-Fc given to mice or monkeys caused a drastic 4-fold increase in Tregs, with nominal changes to CD8 + T cells and NK cells compared with the control.Notably, the increase in Treg numbers could be sustained with continuous mIL-2-Fc treatment over a three-week period, with Treg numbers returning to baseline levels upon cessation of mIL-2-Fc treatment.
The expanded Tregs under mIL-2-Fc treatment demonstrated all the hallmarks of effector Tregs, which are a highly activated and actively dividing Treg subset.They also showed an enhanced suppressive capacity in ex vivo suppression assays.Crucially, a sustained mIL-2-Fc regimen alone was able to provide protection from rejection of minor antigen-mismatched skin grafts, with approximately 60% to 75% of the grafts pendent tolerance, the trimeric high-affinity IL-2R represents an appealing therapeutic target to achieve this goal.
To this end, Efe and collaborators designed an IL-2 mutein fused to the Fc portion of a human IgG1 antibody (termed mIL-2-Fc) (7).Histidine 16 on native IL-2 is structurally essential for the interaction of IL-2 with CD122.Hence, histidine 16 was substituted with a hydrophobic leucine residue.This substitution resulted in a lower affinity of mIL-2-Fc for the dimeric CD122/CD132 IL-2R, while preserving near-native affinity for the trimeric CD25/CD122/CD132 IL-2R.mIL-2-Fc demonstrated a half-life of approximately 9 hours in mice, with nominal detection at 72 hours.Importantly, mIL-2-Fc exhibited specificity for Tregs obtained from either mice, monkeys, or humans.mIL-2-Fc induced downstream IL-2R signaling in both ity for CD25 was reduced compared with native IL-2-Fc, which is an important aspect that was avoided with the IL-2 mutein presented in this issue of the JCI by Efe and colleagues (7).Despite their lower affinities for CD25, in vivo treatment with either IgG-(IL-2N88D) 2 or Fc.Mut24 promoted Tregs and protected from diabetes and graft-versus-host disease (6).However, these Treg-promoting muteins have not been assessed in transplantation settings.

mIL-2-Fc promotes tolerance to nominal graft antigen
Approaches that promote endogenous Tregs provide attractive advantages over adoptive transfer of exogenous Tregs, which are proving costly and time consuming.Given that the field of transplantation is in dire need of new therapeutics that promote Treg-de- [called IgG-(IL-2N88D) 2 and Fc.Mut24].Although these muteins displayed a skewed affinity for CD25 over CD122, their affin-Related Article: https://doi.org/10.1172/JCI173107Conflict of Interest: The authors have declared that no conflict of interest exists.Copyright: © 2024, Camirand et al.This is an open access article published under the terms of the Creative Commons Attribution 4.0 International License.Reference information: J Clin Invest.2024;134(5):e178570. https://doi.org/10.1172/JCI178570.

Figure 1 .
Figure 1.mIL-2-Fc with trimeric IL-2R specificity induces tolerance in low effector T cell contexts.The high-affinity trimeric IL-2R is expressed on Tregs and on recently activated effector T cells among others.mIL-2-Fc with specificity for the trimeric IL-2R, but not the dimeric IL-2R expressed on memory T cells and NK cells, expands Tregs in vivo and induces immunological tolerance to minor antigen-mismatched skin grafts in which low effector T cell levels are present at the time of treatment.In contrast, immunological tolerance with mIL-2-Fc treatment cannot be achieved against major antigen-mismatched skin grafts in which elevated effector T cell levels are present at the time of treatment, despite expansion of Tregs.